Biological material detection element, biological material detection method and apparatus, charged material moving apparatus

ABSTRACT

A biological material detection apparatus which detects a charged biological material such as a gene or protein contained in a sample liquid is disclosed. A biological material detection element includes a substrate, at least one first electrode formed on the substrate, and a plurality of second electrodes which are arrayed at predetermined intervals around the first electrode on the substrate along the circumferential direction and to which ligands that react with predetermined biological materials are respectively immobilized. A sample liquid is introduced toward the first electrode on the substrate. The introduced sample liquid is moved radially toward the second electrodes by electrical control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromthe prior Japanese Patent Applications No. 2001-264696, filed Aug. 31,2001; No. 2001-264716, filed Aug. 31, 2001; and No. 2001-264752, filedAug. 31, 2001, the entire contents of all of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a biological material detectionelement, biological material detection method and apparatus, and chargedmaterial moving apparatus which are used to detect biological materialssuch as genes and proteins.

2. Description of the Related Art

Recently, systems for detecting biological materials such as genes andproteins have been under development. For example, the detection ofgenes is used for the prediction of the curative effect produced byinterferon. A conventional biological material detection technique willbe described with reference to an example of the prediction of curativeeffect produced by interferon.

It is known that when a person is infected with hepatitis C, thisdisease is likely to proceed to hepatic cancer through hepatichepatocirrhosis. One of the medical treatments for the disease is amethod using interferon. It is reported that the injection of interferonhas a curative effect on only about 20 to 30% of the Japanese, andcauses strong side effects even if it produces the curative effect. Forthis reason, attention has recently begun to be paid to personalizedmedical treatment in which the curative effect of interferon ispredicted, and interferon is used only when a curative effect can beexpected.

As a method of predicting the curative effect of interferon, a method ofchecking the type of virus and the amount of virus at the gene level isknown. It is thought that interferon has little effect on type 1b withwhich many Japanese are infected, but does have a curative effect ontype 2a, and that interferon has little effect when the amount of virusis 10⁶ copy/mL or more. In actual diagnoses, these factors are oftenmixed, resulting in difficulty in prediction. Recently, as a method ofpredicting the curative effect of interferon, a method has beenreported, in which single nucleotide polymorphism (SNP) that exists inthe promoter region of gene that codes for M×A protein is used as amarker. According to this report, if the SNP is of type G/G, the effectof interferon is small, whereas if the SNP is of type G/T or T/T,interferon works effectively.

As described above, it is becoming possible to predict the curativeeffect of interferon by analysis at the gene level. All these methodshave used cumbersome, expensive conventional techniques(electrophoresis, Microplate EIA, and the like), and hence moresimplified methods have been required for clinical examination.

Under the circumstances, attention has recently been paid to a geneinspection technique using a biological material detection elementcalled a DNA chip (Beattie et al. 1993, Fodor et al. 1991, Khrapko etal. 1989, and Southern et al. 1994). The DNA chip is formed from aseveral cm square glass or silicon chip on which a plurality of types ofDNA probes with different sequences are immobilized. A mixture of asample gene marked by a fluorescent dye, radiation isotopic element(RI), or the like or non-marked sample gene and marked oligonucleotideis caused to react on the chip. If there is a sequence, in the sample,which is complementary to a DNA probe on the chip, a signal originatingfrom the marker can be obtained at a specific portion on the chip. Ifthe sequences and position of the immobilized DNA probes are known inadvance, a base sequence existing in the sample gene can be easilychecked. Such a DNA chip makes it possible to obtain many kinds ofinformation concerning base sequences by one test, and hence can be usedfor a clinical diagnosis technique (Pease et al. 1994, Parinov et al.1996).

The principle of an electrochemical gene detection method using DNA chipis schematically shown in FIG. 25.

In a typical conventional DNA chip, a sample liquid made of an aqueoussolution of genes is introduced from a sample liquid introductionportion placed on one end portion of the chip surface, flows on variousDNA probes immobilized in the cells of a matrix, and then is dischargedfrom a sample liquid discharge portion placed on the other end portionof the chip surface. The overall DNA chip is covered with a resin case,and the portion where the DNA probes are immobilized is made transparentto read optical signals such as fluorescence.

The above conventional DNA chip is designed such that in the process inwhich a sample liquid flows from one end portion of the chip surface tothe other end portion, the liquid is guided on the DNA probes arrayed inthe form of a matrix. Since it is difficult to make the sample liquiduniformly flow on the DNA probes, it is difficult to make the gene and aDNA probe reliably react with each other. This tends to cause variationsin detection result.

In addition, in general, since the gene concentration in a sample liquidis low, when a conventional DNA chip having no gene concentratingeffect, in particular, a gene to be detected must be amplified inadvance by a gene amplification method such as the PCR method.

Conventionally, in a biological material detection apparatus fordetecting genes by electrochemical measurement using a DNA chip, currentmeasurement is performed while a voltage is applied between electrodesin a proper electrolytic solution stored in a vessel 100, as shown inFIG. 27. Electrodes 101, 102, and 103 called a counterelectrode,reference electrode, and working electrode, are inserted in the vessel100.

The reference electrode 102 is an electrode for applying a referencepotential to the counterelectrode 101, which is held at a predeterminedpotential. A voltmeter 106 is connected between the counterelectrode 101and the reference electrode 102 to measure the potential of thecounterelectrode 101. Besides, a variable DC voltage source 104 isconnected between the counterelectrode 101 and the working electrode103. The variable DC voltage source 104 varies an applied voltagebetween the counterelectrode 101 and the working electrode 103. Thevoltage sweeping causes a current change, which is measured by anammeter 105, thus detecting a gene.

FIG. 28 shows a procedure for detecting a gene by biological materialdetection apparatus having an arrangement like that shown in FIG. 27using a nucleic acid intercalating agent. First of all, a sample liquidis supplied (step S1). With this operation, the sample liquid is made toadhere to a DNA probe (single stranded DNA that reacts with a specificgene) immobilized to the working electrode to convert the DNA in theliquid into a single stranded DNA, thus performing hybridization. Thesample liquid that did not adhere the DNA probe is then cleaned (stepS2). Subsequently, an intercalating reagent (nucleic acid intercalatingagent) that reacts specifically with a double stranded DNA is suppliedto improve the detection sensitivity (step S3), and the unnecessaryintercalating agent is further cleaned (step S4). Finally, a voltage isapplied between the counterelectrode 101 and the working electrode 103,and an oxidation current obtained from the intercalating agent ismeasured, i.e., an electrochemical signal obtained from theintercalating agent is measured (step S5).

A current-potential curve of Hoechst 33258 as a DNA binder is shown inFIG. 26.

As described above, in the conventional biological material detectionapparatus, since current measurement can be performed only once withrespect to one working electrode (DNA chip), it is difficult to improvethe gene detection sensitivity. FIG. 29 shows a change in currentdensity when a given plasmid (pYRB259) is measured by using anelectrochemical DNA chip. As is obvious, since the background current ishigh, a low-concentration gene cannot be detected. In general, since thegene concentration of a sample liquid is low, a target gene must beamplified in advance by a gene amplification method such as the PCRmethod.

BRIEF SUMMARY OF THE INVENTION

The present invention has as its object to provide a biological materialdetection element and detection apparatus which can make a detectiontarget biological material and a ligand reach with each other under auniform condition.

It is another object of the present invention to concentrate abiological material in a sample liquid during detection.

In order to achieve the above objects, according to embodiments of thepresent invention, there is provided a biological material detectionelement which introduces a sample liquid containing a charged biologicalmaterial and detects the biological material, comprising: a substrate;at least one first electrode placed at a position on the substrate towhich the sample liquid is introduced; and a plurality of secondelectrodes which are arrayed at predetermined intervals around the firstelectrode on the substrate in a circumferential direction and to whichligands that react predetermined biological materials are respectivelyimmobilized.

According to embodiments of the present invention, there is provided abiological material detection apparatus which detects a chargedbiological material contained in a sample liquid, comprising: abiological material detection element having a substrate and a pluralityof electrodes which are arrayed at predetermined intervals along acircumferential direction on the substrate and to which ligands thatreact predetermined biological materials are respectively immobilized; asample liquid introduction part to introduce the sample liquid to acentral portion of the array of the electrodes on the substrate; and asample liquid moving mechanism to move the sample liquid introduced tothe central portion on the substrate by the sample liquid introductionpart radially toward the electrodes.

According to embodiments of the present invention, there is provided abiological material detection method of detecting a charged biologicalmaterial contained in a sample liquid with a biological materialdetection element formed by arraying, on a substrate, electrodes towhich ligands that react with predetermined biological materials arerespectively immobilized, the method comprising: after supplying thesample liquid onto the biological material detection element, repeatinga series of steps of (a) supplying an intercalating agent onto thebiological material detection element, (b) measuring an electrochemicalsignal from the intercalating agent which is based on a reaction betweenthe charged biological material and the ligand, and (c) removing theintercalating agent adhering to the ligand, thereby detecting thecharged biological material.

According to embodiments of the present invention, there is provided acharged material moving apparatus which moves a charged material havinga specific charge polarity, comprising: a substrate; a plurality ofelectrodes arrayed on the substrate along a specific direction; and adriving circuit which moves the charged material onto the plurality ofelectrodes along the specific direction by performing driving operationof applying a voltage having an opposite polarity to the charge polarityof the charged material to some of the plurality of electrodes whilesequentially changing a position of an electrode to which the voltagehaving the opposite polarity is to be applied.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus including a biological material detectionelement according to a first embodiment of the present invention;

FIG. 2A is a plan view of an upper holder in the first embodiment;

FIG. 2B is a sectional view of the upper holder in the first embodiment;

FIG. 2C is a bottom view of the upper holder in the first embodiment;

FIG. 3A is a plan view of a lower holder in the first embodiment;

FIG. 3B is a sectional view of the lower holder in the first embodiment;

FIG. 3C is a bottom view of the lower holder in the first embodiment;

FIG. 4 is a plan view of the biological material detection elementaccording to the first embodiment, showing how a driving circuit isconnected to the biological material detection element;

FIGS. 5A to 5C are views for explaining the basic operation of thebiological material detection element according to the first embodiment;

FIGS. 6A to 6C are views for explaining the movement of a detectiontarget biological material on working electrodes in the biologicalmaterial detection element according to the first embodiment;

FIGS. 7A to 7C are views for explaining the electrode arrangement of abiological material detection element according to a modification of thefirst embodiment and the concentration of a detection target biologicalmaterial and its movement to a peripheral portion in the biologicalmaterial detection element;

FIGS. 8A to 8C are views for explaining the movement of a detectiontarget biological material on working electrodes in the biologicalmaterial detection element according to another modification of thefirst embodiment;

FIG. 9 is a plan view showing the arrangement of a biological materialdetection element according to still another modification of the firstembodiment;

FIG. 10 is a plan view of a biological material detection elementaccording to still another modification of the first embodiment, showinghow a driving circuit is connected to the biological material detectionelement;

FIG. 11 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus including a biological material detectionelement according to a second embodiment of the present invention;

FIG. 12 is a flow chart for explaining a biological material detectionprocedure in the second embodiment;

FIG. 13 is a plan view showing the arrangement of a biological materialdetection element according to a modification of the second embodiment;

FIG. 14 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus, to which a charged material movingapparatus is applied, according to a third embodiment of the presentinvention;

FIG. 15 is a plan view showing the schematic arrangement of a biologicalmaterial detection element according to the third embodiment;

FIGS. 16A to 16C are views showing the first example of the drivingoperation of the biological material detection element according to thethird embodiment;

FIGS. 17A to 17C are views showing the second example of the drivingoperation of the biological material detection element according to thethird embodiment;

FIGS. 18A to 18C are views showing the third example of the drivingoperation of the biological material detection element according to thethird embodiment;

FIGS. 19A to 19C are views showing the fourth example of the drivingoperation of the biological material detection element according to thethird embodiment;

FIGS. 20A to 20C are views for explaining another electrode arrangementof a biological material detection element according to the thirdembodiment and the concentration and movement of a detection targetbiological material in the biological material detection element;

FIGS. 21A and 21B are a plan view and sectional view showing stillanother arrangement of a biological material detection apparatusaccording to the third embodiment;

FIG. 22 is a plan view showing the arrangement of a biological materialdetection element according to a modification of the third embodiment;

FIG. 23 is a plan view showing the schematic arrangement of a biologicalmaterial processing apparatus, to which a charged material movingapparatus is applied, according to a fourth embodiment of the presentinvention;

FIG. 24 is a view showing a micellar structure used in a biologicalmaterial processing apparatus, to which a charged material movingapparatus is applied, according to a fifth embodiment of the presentinvention;

FIG. 25 is a view for explaining the principle of an electrochemicalgene detection method;

FIG. 26 is a graph showing an example of a current-potential response ofa DNA binder (Hoechst 33258) in the electrochemical gene detectionmethod;

FIG. 27 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus using conventional electrochemicalmeasurement;

FIG. 28 is a flow chart showing a biological material detectionprocedure based on conventional electrochemical measurement; and

FIG. 29 is a graph showing an example of the gene detection resultobtained by biological material detection using conventionalelectrochemical measurement.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the present invention will be described below withreference to the views of the accompanying drawing.

First Embodiment

FIG. 1 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus including a biological material detectionelement according to a first embodiment of the present invention. A base1 has a protruding element mount portion 2 on its central upper portionon which a biological material detection element 5 (to be described indetail later) is mounted. The base 1 also has a sample liquid passagehole 3 in the two sides in FIG. 1 and a sample liquid outlet 4 in acentral lower portion which communicates with the sample liquid passagehole 3.

An upper holder 6 and lower holder 7 are mounted on the base 1. Theupper and lower holders hold the biological material detection element 5mounted on the element mount portion 2 from both upper and lower sides,and mainly guide a sample liquid into the element 5 and the sampleliquid passing through the element 5 into the sample liquid passage hole3. The structures of the upper and lower holders 6 and 7 will bedescribed in detail later.

A sample liquid introduction portion 8 is formed in the central portionof the upper holder 6. The distal end of a sample liquid supply pipe 10is connected to this sample liquid introduction portion 8. A sampleliquid source (not shown) is connected to the proximal end portion (notshown) of the sample liquid supply pipe 10. A sample liquid receiving aproper positive pressure is introduced from this sample liquid sourceinto the sample liquid introduction portion 8. The sample liquidintroduced into the sample liquid introduction portion 8 is guided ontothe biological material detection element 5 and used for the detectionof a biological material. Thereafter, the sample liquid is dischargedoutside from a sample liquid outlet 4, under a proper negative pressure,through a sample liquid guide duct 9 formed by the lower and upperholders 6 and 7 and the sample liquid passage hole 3 formed in the base1.

A rectangular through hole 11 is formed in the upper holder 6. Thedistal end of a contact electrode 15 can be brought into contact with anelectrode serving as a working electrode on the biological materialdetection element 5 by inserting the contact electrode 15 via thethrough hole 11. By using this contact electrode 15, a biologicalmaterial detection signal can be extracted as an electrical signal suchas a current or potential signal.

FIGS. 2A, 2B, and 2C are a plan view, sectional view, and bottom view ofthe upper holder 6, respectively. Referring to FIG. 2C, the passagewaysof a sample liquid are indicated by the arrows in FIG. 2C. A centralportion 12 (a portion of the base 1 which opposes the element mountportion 2) on the lower surface of the upper holder 6 which opposes thebiological material detection element 5 slightly protrudes downward. Aring-like sample liquid passage hole 13 communicating with the sampleliquid guide duct 9 shown in FIG. 1 is formed around the central portion12. A recess portion 9A that forms the sample liquid guide duct 9 isformed in the lower surface of the upper holder 6.

The sample liquid introduction portion 8 formed in the central portionof the upper holder 6 is a tapered hole. The sample liquid introducedinto this sample liquid introduction portion 8 spreads radially. In thisprocess, the sample liquid is guided onto a ligand immobilizing portionof the biological material detection element 5. Subsequently, the sampleliquid is guided to the sample liquid guide duct 9 through the sampleliquid passage hole 13 and discharged outside from the sample liquidoutlet 4 through the sample liquid passage hole 3 formed in the base 1,as described above.

FIGS. 3A, 3B, and 3C are a plan view, sectional view, and bottom view ofthe lower holder 7, respectively. The lower holder 7 has a hole 14 inits central portion, in which the element mount portion 2 of the base 1shown in FIG. 1 is inserted. Rectangular recess portions 9B and circularholes 9C communicating with the recess portions 9B, which partly formthe other portion of the sample liquid guide duct 9 shown in FIG. 1, areformed in both the left and right sides of the lower holder 7 in FIGS.3A to 3C.

The sample liquid supplied from the sample liquid supply pipe 10 in thismanner is guided by the upper holder 6 and lower holder 7 and introducedfrom the central portion into the biological material detection element5. After the sample liquid is uniformly supplied to the ligandimmobilizing portions formed on the peripheral portion of the element 5,the liquid is discharged from below. Detection of a biological materialin the sample liquid can therefore be done under a uniform condition.

As shown in FIG. 1, the biological material detection element 5 is holdby the upper holder 6 and lower holder 7. In this case, the biologicalmaterial detection element 5 may be immobilized to the biologicalmaterial detection apparatus, i.e., may have an electrode-integratedarrangement. Alternatively, the element 5 may be designed to be detachedfrom the biological material detection apparatus by detaching the upperholder 6, i.e., may have an electrode separation type arrangement.

The detailed arrangement of the biological material detection element 5according to this embodiment will be described next with reference toFIG. 4.

As shown in FIG. 4, the biological material detection element 5 isformed by forming electrodes 21, 22, and 23 and electrode pads 24 on anelement substrate 20. The surfaces of the electrodes 21, 22, and 23 maybe flush with the surface of the element substrate 20, or may beembedded to be slightly recessed from the surface of the elementsubstrate 20. The electrodes 21, 22, and 23 and electrode pads 24 areconnected to each other through, for example, a multilayerinterconnection formed on the element substrate 20.

The circular electrode 21 formed on the central portion functions as acounterelectrode. The electrode 23 formed in an annular shape centeredon the electrode 21 functions as a reference electrode for providing areference potential for the counterelectrode. The circular electrodes 22are arranged at a predetermined pitch on a circumference on the innerside of the electrode 23. These electrodes 22 function as workingelectrodes for detecting a biological material. The surfaces of theelectrodes 21, 22, and 23 are covered with a thin insulating film (notshown), and this thin insulating film is subjected to lithographyprocessing. In the lithography processing, parts of the surfaces of theelectrode 21 and 23 are removed, and the conductive portions of theelectrodes are exposed so that electrical signals can be taken outthrough the parts. The details of the lithography processing carried outon the thin insulating film will be described later.

At least one specific detection ligand is immobilized to the electrode22 serving as a working electrode. That is, the electrode 22 also servesas a ligand immobilizing portion. The ligand immobilized to eachelectrode 22 is selected from one of, for example, a gene, gene probe,protein, protein segment, coenzyme, receptor, and sugar chain inaccordance with the biological material to be detected.

If different ligands are immobilized to the respective electrodes 22, aplurality of biological materials can be detected at once. In addition,if identical ligands are immobilized to the respective electrodes 22,many biological materials can be detected at once. If many electrodes 22(ligand immobilizing portions) are patterned on the element substrate 20in advance by photolithography, the productivity of biological materialdetection elements 5 improves.

If a biological material to be detected is a gene, a DNA probe isimmobilized as a ligand to the electrode 22. As is known, a DNA probe isa single stranded gene that reacts with a specific gene. If genes in asample liquid are converted into a single stranded gene in advance, onlya gene having a specific sequence in correspondence with the DNA probeimmobilized to the electrode 22 is trapped by the electrode 22.Subsequently, the DNA probe and the gene are complementarily bound toeach other (hybridization).

The above arrangement will be described in further detail. First of all,a substrate material used for the element substrate 20 is, but is notlimited to, an inorganic insulating material such as glass, silicaglass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, orsilicon nitride, for example. Alternatively, one of the followingorganic materials can be used as a substrate material: polyethylene,ethylene, polypropylene, polyisobutylene, polymethylmethacrylate,polyethylene terephthalate, unsaturated polyester, fluorine-containingresin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate,polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile,polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urearesin, epoxy resin, melamine resin, a styrene-acrylonitrile copolymer,an acrylonitrile-butadiene-styrene copolymer, silicone resin,polyphenylene oxide, polysulfone, and the like. In addition, ifbiological material detection is to be performed by the optical methodto be described later, a thin fiber film such as nylon or cellulose canbe used.

The electrode materials to be used for the electrodes 21, 22, and 23 arenot specifically limited. As a material (including ligand immobilizingspot) for the electrode 22 serving as a working electrode, inparticular, when biological material detection is to be performedelectrochemically, one of the following materials can be used: a singlemetal such as gold, gold alloy, silver, platinum, mercury, nickel,palladium, silicon, germanium, gallium, or tungsten, alloys eachcontaining at least two of these metals, carbon such as graphite orglassy carbon, oxides thereof, compounds thereof, semiconductorcompounds such as silicon oxide, and various kinds of semiconductordevices such as a CCD, FET, and CMOS, for example.

As a method of forming the electrodes 21, 22, and 23, plating, printing,sputtering, vapor deposition, or the like can be used. As a vapordeposition method, one of a resistance-heating method, RF heatingmethod, and electron beam heating method can be used. As a sputteringmethod, one of DC bipolar sputtering, bias sputtering, asymmetrical ACsputtering, getter sputtering, and RF sputtering can be used. For anelectrode, an electrolytic polymer film or conductive high polymer suchas polypyrrole or polyaniline can be used.

An insulating material used for a thin insulating film that covers thesurfaces of the electrodes 21, 22, and 23 is, but is not limited to,photopolymer or photoresist material, for example. As a photoresistmaterial, an exposure photoresist, far ultraviolet photoresist, X-rayphotoresist, or electron beam photoresist may be used. A main materialfor an exposure photoresist includes cyclized rubber, polycinnamic acid,and novolac resin. As a far ultraviolet photoresist, cyclized rubber,phenol resin, polymethylisopropenylketone (PMIPK),polymethylmethacrylate (PMMA), or the like may be used. As an X-rayphotoresist, a material written in “Thin Film Handbook” (Ohmsha, Ltd.)as well as a COP and metal acrylate. As an electron beam resist, amaterial written in “Thin Film Handbook” (Ohmsha, Ltd.) such as PMMA canbe used. The resist to be used in this case preferably has a thicknessof 10 nm or more and 1 mm or less.

The area of the electrode 22 serving as a working electrode can be madeuniform by covering the electrode 22 with a photoresist and performinglithography. With this process, the amounts of ligand such as DNA probeto be immobilized become uniform among the electrodes 22. This makes itpossible to perform biological material detection with excellentreproducibility. Conventionally, a resist material is generally removedin the end. If, however, the electrode 22 is used for the detection of agene immobilized to a DNA probe, the resist material can be used as partof the electrode 22 without being removed. In this case, a materialhaving high water resistance must be used as a resist material.

For the thin insulating film to be formed on the electrodes 21, 22, and23, a material other than photoresist materials can be used. Forexample, oxides, nitrides, and carbides of Si, Ti, Al, Zn, Pb, Cd, W,Mo, Cr, Ta, Ni, and the like and alloys thereof can be used. After athin film is formed by sputtering, vapor deposition, CVD, or the likeusing one of these materials, the film is patterned by photolithographyto form exposed electrode portions, thus controlling the area constant.

These electrodes 21, 22, and 23 are connected to a driving circuit 25via the electrode pads 24. The driving circuit 25 applies voltages withpredetermined polarities to the respective electrodes 21, 22, and 23 todisperse the sample liquid, introduced onto the central portion on thebiological material detection element 5, to the surroundings radially,guide the liquid onto a given electrode 22 serving as a workingelectrode, and sequentially move a detection target biological materialin the sample liquid on the electrode 22 in the array direction(circumferential direction) of the electrodes 22.

This operation will be described below with reference to FIGS. 5A to 5C.When a detection target biological material is a gene, a sample liquidwhich is an aqueous solution of genes is introduced from the sampleliquid supply pipe 10 onto the biological material detection element 5via the sample liquid introduction portion 8 and supplied onto theelectrode 21 on the central portion. The charge polarity of the gene isnegative.

As shown in FIG. 5A, when the sample liquid is to be introduced, thedriving circuit 25 applies a negative voltage whose polarity is the sameas the charge polarity of the gene to the electrode 21 serving as acounterelectrode, a positive voltage whose polarity is opposite to thecharge polarity of the gene to the electrodes 22 serving as workingelectrodes, and a negative voltage to the electrode 23 as a referenceelectrode. The sample liquid supplied onto the electrode 21 receiveselectrostatic repulsive force from the electrode 21 to which thenegative voltage whose polarity is the same as the charge polarity ofthe gene is applied, and moves radially toward the peripheral portion.In this case, when a slight negative pressure is applied to the sampleliquid, the sample liquid moves more quickly.

The sample liquid that has moved from the electrode 21 toward theperipheral portion will reach the electrode 22. In this case, since apositive voltage whose polarity is opposite to the charge polarity ofthe genes in the sample liquid is applied, the gene is trapped on theelectrode 22 by electrostatic attracting force. In this case, since anegative voltage whose polarity is the same as the charge polarity ofthe gene is applied to the ring-like electrode 23 serving as a referenceelectrode placed on the outer circumferential side of the electrodes 22,like the electrode 21, the gene on the electrode 22 receiveselectrostatic attracting force originating from the electrode 23 to beconfined and does not move outside from the electrode 23.

When a gene in the sample liquid is trapped on the electrode 22 in thismanner, the DNA probe which is the ligand immobilized to the electrode22 and the specific gene in the sample liquid react and bind with eachother. This is hybridization. In this case, the gene on the electrode 22is concentrated when it is confined by electrostatic attracting forceoriginating from the electrode 23, as described above, and hence thegene efficiently reacts with the ligand, i.e., hybridization isefficiently performed.

As shown in FIG. 5B, the polarity of only the voltage applied to theelectrode 23 is changed to positive polarity which is opposite to thecharge polarity of the gene while the polarities of the voltages appliedto the electrodes 21 and 22 are kept the same as those shown in FIG. 5A.With this operation, of the genes in the sample liquid on the electrode22, genes that did not contribute to reaction with the ligand areseparated from the electrode 22 by the electrostatic attracting forceoriginating from the electrode 23 and trapped on the electrode 23.

When voltages with negative polarity are applied to both the electrode22 and the electrode 23, the genes that have been trapped on theelectrode 23, i.e., that have not contributed to reaction with theligand, are further moved outward and discharged from the sample liquidoutlet 4, together with the sample liquid on the element substrate 20,via the sample liquid guide duct 9 and sample liquid passage hole 3.

The polarities of the electrodes 21, 22, and 23 may change from thestate shown in FIG. 5A to that of FIG. 5C, not to that of FIG. 5B.Specifically, as shown in FIG. 5C, the polarity of the voltage appliedto the electrode 21 is inverted to positive, the polarity of the voltageapplied to the electrode 22 is inverted to negative, and the polarity ofthe voltage applied to the electrode 23 is inverted to positive. Notethat, in a modification of this embodiment, the polarity of theelectrode 21 may be negative in FIG. 5C. Since the electrode 21 haspositive polarity, there will result a more enhanced effect ofseparating the gene entangled on the electrode 22 from it. However, someunnecessary genes still remain in a central portion and it will benecessary to wash them off later in a separate step. If the electrode 21has negative polarity, these unnecessary genes entangled on theelectrode 22 are attracted towards the electrode 23 located on an outerside. Thus, the washing step that has to be carried out later isfacilitated. It is preferable that the polarity of the electrode 21,whether it should be set to positive or negative, be determined inconsideration of the design of the overall system including the washingstep. In the case shown in FIG. 5C as well, it is possible to removeuncombined genes from the electrode 22 and thereby perform thehybridization with a high degree of efficiency.

According to the above description of the operation, as shown in FIGS.5A and 5B, voltages whose polarity (negative polarity) is the same asthe charge polarity of the detection target gene are applied to all theelectrodes 22. Through the use of the arrangement in which theelectrodes 22 are arrayed on the circumference, a gene in a sampleliquid may be sequentially moved on the electrodes 22 in the arraydirection (circumferential direction) by dynamically switching thepolarity of the voltage applied to the electrodes 22.

This operation will be described below with reference to FIGS. 6A to 6C.As shown in FIG. 6A, positive voltages are applied to pairs of adjacentelectrodes, of the electrodes 22, which are enclosed with the dottedlines, and negative voltages are applied to the electrodes adjacent tothe pairs. The polarities of the voltages applied to the electrodes 22are arranged like“positive—positive—negative—positive—positive—negative—positive—positive—negative—positive. . . ” when viewed from the circumferential direction, i.e., the arraydirection of the electrodes 22.

After an elapse of a predetermined unit time, as shown in FIG. 6B, theposition of each pair of electrodes to which positive voltages are to beapplied is shifted by one electrode, and the position of each electrodewhich is adjacent to each pair of electrodes and to which a negativevoltage is to be applied is shifted by one electrode accordingly. Whenthe predetermined unit time has further elapsed, the position of eachpair of electrodes to which positive voltages are to be applied and theposition of each electrode to which a negative voltage is to be appliedare shifted by one electrode, as shown in FIG. 6C. In the case shown inFIGS. 6B and 6C, the position of each pair of electrodes to whichpositive voltages are to be applied and the position of each electrodeto which a negative voltage is to be applied are shifted clockwise.Subsequently, such switching of the polarities of applied voltages willbe done every unit time, i.e., in a predetermined cycle.

By applying voltages to the electrodes 22 while switching the polaritiesin this manner, a detection target biological material (e.g., a gene) ina sample liquid is moved on the array of the electrodes 22 in thecircumferential direction. This allows the biological material uniformlyand efficiently reacts to a ligand (e.g., a DNA probe) immobilized toeach electrode 22. That is, in the process of moving on the array of theelectrodes 22, the detection target biological material is located,without fail, on the electrode to which the ligand having acomplementary relationship with the detection target biological materialis immobilized, and can react with the ligand.

In this case, since materials nonspecifically binding with electrodes,of the electrodes 22, to which voltages having the same polarity as thatcharge polarity of the detection target biological material are appliedare forcibly removed, the detection precision of the detection targetbiological material (a gene in this case) trapped on the electrode 22 asa ligand immobilizing portion can be greatly improved.

In the case shown in FIGS. 6A to 6C, voltages having the first polarity(negative polarity in the above case) are applied to each pair ofadjacent electrodes of the electrodes 22, and a voltage having thesecond polarity (positive polarity in the above case) opposite to thefirst polarity is applied to each electrode adjacent to each pair ofelectrodes while the positions of electrodes to which such voltages areapplied are shifted by one electrode at a time in the circumferentialdirection of the electrodes 22. However, the present invention is notlimited to this. Letting n be the number of electrodes to which voltageshaving the first polarity are applied, m be the number of electrodes towhich voltages having the second polarity are applied, and p be thenumber of electrodes by which the voltage application positions areshifted when the polarities of applied voltages are switched, n, m, andp can take arbitrary numbers equal to or larger than one. Most simply,it suffices if n=m=p=1. In this case, the polarity of an applied voltageis periodically and alternately switched between positive polarity andnegative polarity from the viewpoint of one electrode 22.

The electrode arrangement of a biological material detection elementaccording to a modification of the first embodiment will be describednext with reference to FIGS. 7A to 7C.

In the biological material detection element shown in FIGS. 7A to 7C, acircular electrode 31 functioning as a counterelectrode is placed on thecentral portion, and an annular electrode 34A is placed on the outercircumferential side of the electrode 31 at a predetermined distance.Another annular electrode 34B is placed on a circumference located onthe outer circumferential side of the electrode 34A at a predetermineddistance. As in the above embodiment, a plurality of circular electrodes32 are arrayed at a predetermined pitch. These electrodes 32 function asworking electrodes. In addition, annular electrodes 33A and 33B aresequentially arranged on the outer circumferential side of theelectrodes 32 at predetermined distances. These electrodes 33A and 33Bfunction as reference electrodes.

In this embodiment, a detection target biological material in a sampleliquid can be concentrated and moved on a peripheral portion byswitching the polarities of voltages applied to the electrodes 31, 34A,34B, 32, 33A, and 33B using a driving circuit (not shown), as shown inFIGS. 7A to 7C.

First of all, as shown in FIG. 7A, a negative voltage, positive voltage,and negative voltage are respectively applied to the electrode 31,electrode 34A, and electrode 34B. In addition, positive voltages areapplied to the electrodes 32, and negative voltages are applied to theelectrodes 33A and 33B. In this case, as in the above embodiment, adetection target biological material (e.g., a gene) with negative chargepolarity in a sample liquid introduced onto the central portion of thebiological material detection element moves to the peripheral portiondue to the electrostatic repulsive force originating from the centralelectrode 31 to which a voltage having the same polarity as the chargepolarity is applied.

In this case, a voltage having an opposite polarity to the chargepolarity of the detection target biological material is applied to theelectrode 34A located closer to the outer circumferential side than theelectrode 31, and a voltage having the same polarity as the chargepolarity is applied to the electrode 34B located closer to the outercircumferential side than the electrode 34A. With this operation, thedetection target biological material that have moved from the electrode31 onto the electrode 34A is trapped and concentrated on the electrode34A due to the electrostatic attracting force produced by the electrode34A and the electrostatic repulsive force produced by the electrode 31and electrode 34B located on the two sides of the electrode 34A.

The polarity of the voltage applied to the electrode 34A and thepolarity of the voltage applied to the electrode 34B are then invertedto negative polarity and positive polarity, respectively, by the drivingcircuit, as shown in FIG. 7B. The polarities of the voltages applied tothe remaining electrodes 31, 32, 33A, and 33B are kept the same as thoseshown in FIG. 7A. The detection target biological material trapped onthe electrode 34A in the state shown in FIG. 7A moves onto the electrode34B due to the electrostatic repulsive force produced by the electrode34A and the electrostatic attracting force produced by the electrode 34Bin the state shown in FIG. 7B.

When the polarity of only the voltage applied to the electrode 34B inthe state shown in FIG. 7B is inverted to negative polarity by thedriving circuit as shown in FIG. 7C, the detection target biologicalmaterial that has moved onto the electrode 34B moves onto the electrode32 due to the electrostatic repulsive force produced by the electrodes34A and 34B and the electrostatic attracting force produced by theelectrode 32. In this state, the detection target biological material onthe electrode 32 is trapped and concentrated on the electrode 32 due tothe electrostatic attracting force produced by the electrode 32 and theelectrostatic repulsive force produced by the electrodes 34A and 33A.

In this manner, the detection target biological material in the sampleliquid introduced onto the central portion of the biological materialdetection element is sequentially concentrated and moved to theperipheral portion. Finally, the detection target biological materialcan be supplied in a concentrated state onto the electrode 32 on whichthe ligand is immobilized. According to this embodiment, since thedetection target biological material is concentrated on the electrode 32to which the ligand is immobilized, the detection target biologicalmaterial and the ligand can be made to react with each other efficientlywithout amplifying the detection target biological material in advanceby a gene amplification method such as the PCR method. This improves thedetection efficiency.

In the biological material detection element according to anothermodification of the first embodiment, as in the above embodiment, a genein a sample liquid may be sequentially moved on the electrodes 32 in thearray direction (circumferential direction) by dynamically switching thepolarity of a voltage applied to each electrode 32 serving as a workingelectrode, as shown in FIGS. 8A to 8C.

First of all, as shown in FIG. 8A, positive voltages are applied to eachpair of adjacent electrodes, of the electrodes 32, which are enclosedwith the dotted line, and a negative voltage is applied to eachelectrode adjacent to each pair of adjacent electrodes. After an elapseof a predetermined unit time, as shown in FIG. 8B, the position of eachpair of adjacent electrodes to which positive voltages are to be appliedis shifted by one electrode, and the position of each electrode adjacentto each pair of adjacent electrodes by one electrode accordingly. Whenthe predetermined unit time has further elapsed, the position of eachpair of adjacent electrodes to which positive voltages are to be appliedand the position of each electrode to which a negative voltage is to beapplied are shifted by one electrode, as shown in FIG. 8C. Subsequently,the polarities of applied voltages are switched every unit time, i.e.,in a predetermined cycle, to allow the detection target biologicalmaterial in the sample liquid to move on the array of electrodes 32 inthe circumferential direction and efficiently react with the ligandimmobilized to each electrode 32.

FIG. 9 shows the arrangement of a biological material detection elementaccording to still another embodiment of the present invention.

In the biological material detection element according to thisembodiment, a first electrode 41 having a circular shape and functioningas a counterelectrode is placed on a central portion on an elementsubstrate 40, and a third electrode 43 having an annular shape andfunctioning as a reference electrode is placed on a peripheral portionas in the above two embodiments. This embodiment differs from the aboveembodiments in that second electrodes 42A and 42B functioning as workingelectrodes are arrayed on two concentric circumferences. In this case,electrodes serving as working electrodes are formed in two arrays.However, three or more arrays of electrodes may be formed.

By forming a plurality of arrays of electrodes serving as workingelectrodes in this manner, the ligand immobilized to each electrode anda detection target biological material can be made to reach with eachother more reliably, thus further improving the detection efficiency.

FIG. 10 is a plan view of a biological material detection elementaccording to still another modification of the first embodiment showinghow a driving circuit is connected to the biological material detectionelement. A biological material detection element 5 according to thisembodiment, the first electrode 21 described in the above embodiment isomitted, and a position where the electrode 21 is removed, i.e., thecentral portion of the array of second electrodes 22, serves as a sampleliquid receiving portion 25. In addition, as the electrode 21 isremoved, the interconnections between the electrode 21 and a drivingcircuit 5 are removed.

In the above embodiment, as described with reference to, for example,FIG. 5A, when a negative voltage whose polarity is the same as thecharge polarity of a gene is applied to the first electrode 21, and apositive voltage whose polarity is opposite to the charge polarity ofthe gene is applied to each second electrode 22, the sample liquidsupplied onto the electrode 21 receives the electrostatic repulsiveforce produced by the charge polarity of the gene and the electrode 21.This makes it easy to move the sample liquid radially toward theelectrodes 22 on the peripheral portion.

If a negative pressure applied to the sample liquid is increased to acertain degree when the sample liquid is to be discharged outside fromthe sample liquid outlet 4 described above, the sample liquid suppliedto the sample liquid receiving portion 25 can be moved toward theelectrode 22 side by only the electrostatic attracting force produced bythe second electrodes 22 without using the electrostatic repulsive forceproduced by the first electrode 21 as in the above embodiment.

In addition, a similar modification can also be applied to thearrangements shown in FIGS. 7A to 9. For example, the first electrode 31in FIGS. 7A to 8C or the first electrode 41 in FIG. 9 may be removed,and a sample liquid receiving portion can be formed at the positionwhere the electrode 31 or 41 is removed.

A sample to be processed by the biological material detection element orbiological material detection apparatus according to the embodiment maybe, but is not limited to, for example, blood, blood serum, leukocyte,urine, stool, sperm, saliva, tissue, cultured cell, expectoration, andthe like. If a detection target biological material is a gene, the geneis extracted from these samples. The extraction method may be, but isnot limited to a liquid-liquid extraction method such as aphenol-chloroform method or a solid liquid extraction method using acarrier. Alternatively, a commercially available nucleic acid extractionmethod such as QIAamp (available from QIAGEN), SUMAI test (availablefrom Sumitomo Metal Industries, Ltd.) can be used.

The gene sample solution extracted in this manner is then introducedonto the biological material detection element (DNA chip) described inthe above embodiment, and a hybridization reaction is caused on anelectrode to which a DNA probe as a ligand is immobilized. A bufferingsolution that falls within the range of ion intensities of 0.01 to 5 andthe range of pH5 to pH10 is used as a reaction solution. A hybridizationaccelerating agent such as dextran sulfate, salmon sperm DNA, bovinethymus DNA, EDTA, or surfactant can be added to this solution, asneeded. The extracted sample gene is added to this solution. Thesolution must be heat-denatured at 90° C. or more before introductioninto the biological material detection element. An unreacted sample genecan be recovered from the sample liquid outlet 4 to be introduced intothe biological material detection element again, as needed.

The extracted gene can be detected by marking in advance it with afluorescent dye such as FITC, Cy3, Cy5, or rhodamine, an enzyme such asbiotin, hapten, oxidase, or phosphatase, or an electrochemically activematerial such as ferrocene or quinones, or by using a second probemarked with such a material. If the gene is marked with a fluorescentdye, it can be optically detected.

When the gene is to be detected by using an electrochemically active DNAbinder, detection is done by the following procedure.

A DNA binder which selectively combines with a double stranded DNAportion is made to react with the double stranded DNA portion formed onthe surface of an electrode (working electrode) to which a DNA probe isimmobilized, thereby performing electrochemical measurement. The DNAbinder to be used in this case is, but is not limited to, for example,Hoechst 33258, acridine orange, quinaccrine, downomycin,metallointercalator, bis-intercalator such as bis-acridine,tris-intercalator, or polyintercalator. In addition, such anintercalator can be modified in advance by an electrochemically activemetal complex such as ferrocene or viologen.

Although the concentration of a DNA binder varies depending on the typeof material, the material is generally used within the range of 1 ng/mLto 1 mg/mL. In this case, a buffering agent within the range of ionintensities of 0.001 to 5 and the range of pH5 to pH10 is used.

After an electrode serving as a working electrode and a DNA binder aremade to react with each other, cleaning is performed, andelectrochemical measurement is performed. Electrochemical measurement isperformed by a triple electrode type including a reference electrode,counterelectrode, and working electrode or a double electrode typeincluding a counterelectrode and working electrode. In measurement, apotential equal to or higher than a potential at which the DNA binderreacts is applied, and a reaction current value originating from the DNAbinder is measured. In this case, the potential is swept at a constantvelocity or pulses or a constant potential can be applied. Inmeasurement, currents and voltages are controlled by using apotentiostat, digital multimeter, function generator, and the like.

EXAMPLE 1

The biological material detection element described with reference toFIGS. 1 to 3C and 7A to 8C was formed into an electrode-integratedinterferon curative effect predicting DNA chip and the followingexperiment was conducted.

First of all, a chromosome DNA was extracted from human leukocyte, a M×Agene fragment of about 100 bp was PCR-amplified by using a properprimer. The amplified fragment was heat-denatured. The resultantfragment was then introduced into the DNA chip. Note that a DNA probeassociated with SNP (single nucleotide polymorphism) existing in the M×Agene was immobilized in advance on an electrode 32 of the DNA chip.After the sample was introduced, it was left standing for two hr, andcleaning was done with a buffering agent. When a DNA binder (Hoechst33258) was made to act, it was found that an interferon curative effectcould be predicted even if the charge was not electrically controlled.However, it became clear that the results vary depending on the mannerin which the liquid flowed.

In contrast to this, it was found that when the introduced gene wasmoved to the peripheral portion while being concentrated by switchingthe polarities of voltages applied to the respective electrodes, asshown in FIGS. 8A to 8C, and the gene was moved on the array ofelectrodes 32 by switching the polarities of voltages applied to therespective electrodes 32 while the gene was trapped on the electrode 32,as shown in FIGS. 7A to 7C, a curative effect could be accuratelypredicted without performing PCR amplification.

EXAMPLE 2

A biological material detection element similar to that described in theembodiments was formed into an electrode separate type biologicalmaterial detection chip, and the following experiment was conducted.

Antibodies for various human tumor markers were immobilized to a nylonfilm in advance. Electrodes were then arranged under the film so as tobe in contact therewith. When a tumor marker was detected by using humanserum as a sample, it was found that the marker could be detected withhigh reproducibility and high sensitivity on the order of 0.1 ng/mL. Inthis case, an antibody marked with horseradish peroxidase was used asthe second antibody, and a framework for luminescence detection wasused.

Second Embodiment

FIG. 11 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus including a biological material detectionelement according to a second embodiment of the present invention. Abase 1 has a protruding element mount portion 2 on its central upperportion on which a biological material detection element 5 is mounted.The base 1 also has a liquid passage hole 3 in the two sides in FIG. 1which allows a sample liquid, intercalating agent, cleaning solution, orthe like to pass through and a liquid outlet 4 in a central lowerportion which communicates with the liquid passage hole 3. An upperholder 6 and lower holder 7 are mounted on the base 1. The upper andlower holders hold the biological material detection element 5 mountedon the element mount portion 2 from both upper and lower sides, andmainly guide a liquid into the element 5 and the liquid passing throughthe element 5 into the liquid passage hole 3. A liquid introductionportion 8 is formed in the central portion of the upper holder 6. Thedistal end of a liquid supply pipe 10 is connected to this liquidintroduction portion 8. One of a sample liquid supply section 51,intercalating agent supply section 52, and cleaning solution supplysection 53 which are controlled by a control section 50 is selectivelyconnected to the liquid supply pipe 10. A measuring section 54controlled by the control section 50 measures an electrochemical signalby using an intercalating agent and outputs the detection result on abiological material, as will be described later. The liquid introducedfrom the liquid supply pipe 10 into the liquid introduction portion 8 isguided onto the biological material detection element 5 and used for thedetection of a biological material. Thereafter, the liquid is dischargedoutside from a liquid outlet 4 through a liquid guide duct 9 formed bythe lower and upper holders 6 and 7 and the liquid passage hole 3 formedin the base 1. A rectangular through hole 11 is formed in the upperholder 6. The distal end of a contact electrode 15 can be brought intocontact with an electrode serving as a working electrode on thebiological material detection element 5 by inserting the contactelectrode 15 via the through hole 11. By using this contact electrode15, a biological material detection signal can be extracted as anelectrical signal such as a current or potential signal.

In the biological material detection apparatus shown in FIG. 11, thedetailed arrangements of the upper and lower holders 6 and 7 are thesame as those shown in FIGS. 2A to 3C. The detailed arrangement of thebiological material detection element 5 is also the same as that shownin FIG. 4. As described above, when a detection target biologicalmaterial is a gene, a DNA probe is immobilized as a ligand to anelectrode 22. The genes in the sample liquid are converted into a singlestranded gene in advance. Only a gene having a specific sequence istrapped on the electrode 22 in correspondence with the DNA probeimmobilized to the electrode 22. Subsequently, the DNA probe and genecomplementarily combine with each other (hybridization). Furthermore,the same materials and methods as those described above with referenceto FIG. 4 are used as a substrate material used for an element substrate20, electrode materials used for an electrode 21, the electrodes 22, andan electrode 23, methods of forming the electrodes 21, 22, and 23, aninsulating material used for a thin insulating film that covers thesurfaces of the electrodes 21, 22, and 23, and the like.

A procedure for detecting a biological material in this embodiment willbe described below with reference to the flow chart of FIG. 12.

First of all, the sample liquid supply section 51 supplies a sampleliquid containing a detection target biological material to the liquidsupply pipe 10 under the control of the control section 50 (step S1).The sample liquid supplied to the liquid supply pipe 10 is introducedonto the biological material detection element 5 via the liquidintroduction portion 8 and moved from the electrode 21 to the electrodes22 and electrode 23 (see FIG. 4) by the electrostatic force produced bythe electrodes 21 to 23 to which predetermined voltages are applied froma driving circuit 25. Finally, the sample liquid is separated from thebiological material detection element 5 and discharged from the liquidoutlet 4 via the liquid guide duct 9 and liquid passage hole 3.

In this process, the sample liquid adheres to the ligand immobilized tothe electrode 22, and hybridization is performed. More specifically,when, for example, a detection target biological material is a gene, anda ligand is a DNA probe, i.e., a single stranded DNA that reacts with aspecific gene, the DNA in the sample liquid is converted into singlestranded DNA when the sample liquid adheres to the DNA probe.

The first cleaning is then performed (step S2). In this first cleaningstep, the cleaning solution supply section 53 supplies a cleaningsolution to the liquid supply pipe 10 under the control of the controlsection 50. With this operation, an unnecessary sample liquid in thebiological material detection apparatus, and more specifically, a sampleliquid other than the sample liquid (biological material) adhering tothe electrode 22 is cleaned and removed. The removed unnecessary sampleliquid is discharged, together with the cleaning solution.

Subsequently, the intercalating agent supply section 52 supplies anintercalating agent for improving the detection sensitivity of abiological material, e.g., a nucleic acid intercalating agent thatspecifically reacts to double stranded DNA, to the liquid supply pipe 10under the control of the control section 50 (step S3). The secondcleaning is then performed (step S4).

In the second cleaning step, the cleaning solution supply section 53supplies a cleaning solution to the liquid supply pipe 10 under thecontrol of the control section 50. With this operation, an unnecessarysample liquid, and more specifically, a sample liquid other than thesample liquid adhering to the electrode 22 is cleaned and removed. Theremoved unnecessary sample liquid is discharged, together with thecleaning solution, via the cleaning solution discharge route in thesample liquid supply processing in step S1.

The measuring section 54 applies a voltage between the electrode 21 as acounterelectrode and the electrode 22 as a working electrode under thecontrol of the control section 50. The measuring section 54 measures acurrent flowing via the two electrodes 21 and 22, i.e., an oxidationcurrent that enters the bond portion between the ligand immobilized tothe electrode 22 and the specific biological material and is obtainedfrom an intercalating agent adhering to the bond portion, therebymeasuring an electrochemical signal by using the intercalating agent(step S5).

The third cleaning step is performed (step S6). In the third cleaningstep, the cleaning solution supply section 53 supplies a cleaningsolution to the liquid supply pipe 10 under the control of the controlsection 50. In this case, all the intercalating agent including theintercalating agent that adheres to the ligand immobilized to theelectrode 22 and has contributed to the measurement of theelectrochemical signal is cleaned and removed.

Of steps S1 to S6, steps S3 to S6 are repeatedly executed until it isdetermined in step S7 that the number of times of execution reaches apredetermined number of times. That is, a series of steps, i.e., (a)supply of an intercalating agent, (b) current measurement (measurementof electrochemical signal by the intercalating agent), and (c) removalof the intercalating agent adhering to the ligand, is repeated.

The set traffic capacity 34 integrates a plurality of measurementresults obtained in this series of steps, i.e., current (oxidationcurrent) values dependent on a detection target biological material.With this operation, only electrochemical signals originating from theintercalating agent dependent on the detection target biologicalmaterial are integrated, and random noise components such as backgroundcurrents are canceled in the process of integration. This makes itpossible to detect a specific biological material with high sensitivity.

FIG. 13 is a plan view showing the arrangement of a biological materialdetection element according to a modification of the second embodiment.

In the biological material detection element according to thisembodiment, a liquid introduction portion 41 is formed at one endportion (the left end in FIG. 13) on an element substrate 40, and aliquid discharge portion 43 is formed at the other end (the right end inFIG. 13), and electrodes 42 serving as working electrodes are arrayed inthe central portion in the form of a matrix. A sample liquid,intercalating agent, or cleaning solution is introduced from the liquidintroduction portion 41. This liquid is discharged from the liquiddischarge portion 43 after moving on the electrodes 42.

Even with the use of a biological material detection element having suchan arrangement, the same effects as those of the above embodiment can beobtained by performing the steps in the procedure shown in FIG. 12.

A sample to be processed by the biological material detection element orbiological material detection apparatus according to the embodiment is,but is not limited to, for example, blood, blood serum, leukocyte,urine, stool, sperm, saliva, tissue, cultured cell, expectoration, andthe like. If, for example, a detection target biological material is agene, the gene is extracted from these samples. An extraction method isnot specifically limited. A liquid-liquid extraction method such as aphenol-chloroform method or a solid liquid extraction method using acarrier can be used. Alternatively, a commercially available nucleicacid extraction method such as QIAamp (available from QIAGEN), SUMAItest (available from Sumitomo Metal Industries, Ltd.) can be used.

The gene sample solution extracted in this manner is then introducedonto the biological material detection element (DNA chip) described inthe above embodiment, and a hybridization reaction is caused on anelectrode to which a DNA probe as a ligand is immobilized. A bufferingsolution that falls within the range of ion intensities of 0.01 to 5 andthe range of pH5 to pH10 is used as a reaction solution. A hybridizationaccelerating agent such as dextran sulfate, salmon sperm DNA, bovinethymus DNA, EDTA, or surfactant can be added to this solution, asneeded. The extracted sample gene is added to this solution. Thesolution must be heat-denatured at 90° C. or more before introductioninto the biological material detection element. An unreacted sample genecan be recovered from the sample liquid outlet 4 to be introduced intothe biological material detection element again, as needed.

The extracted gene can be detected by marking in advance it with afluorescent dye such as FITC, Cy3, Cy5, or rhodamine, an enzyme such asbiotin, hapten, oxidase, or phosphatase, or an electrochemically activematerial such as ferrocene or quinones, or by using a second probemarked with such a material. If the gene is marked with a fluorescentdye, it can be optically detected.

When the gene is to be detected by using an electrochemically active DNAbinder, detection is done by the following procedure.

A DNA binder which selectively combines with a double stranded DNAportion is made to react with the double stranded DNA portion formed onthe surface of an electrode (working electrode) to which a DNA probe isimmobilized, thereby performing electrochemical measurement. The DNAbinder to be used in this case is, but is not limited to, for example,Hoechst 33258, acridine orange, quinaccrine, downomycin,metallointercalator, bis-intercalator such as bis-acridine,tris-intercalator, or polyintercalator. In addition, such anintercalator can be modified in advance by an electrochemically activemetal complex such as ferrocene or viologen.

Although the concentration of a DNA binder varies depending on the typeof material, the material is generally used within the range of 1 ng/mLto 1 mg/mL. In this case, a buffering agent within the range of ionintensities of 0.001 to 5 and the range of pH5 to pH10 is used.

After an electrode serving as a working electrode and a DNA binder aremade to react with each other, cleaning is performed, andelectrochemical measurement is performed. Electrochemical measurement isperformed by a triple electrode type including a reference electrode,counterelectrode, and working electrode or a double electrode typeincluding a counterelectrode and working electrode. In measurement, apotential equal to or higher than a potential at which the DNA binderreacts is applied, and a reaction current value originating from the DNAbinder is measured. In this case, the potential is swept at a constantvelocity or pulses or a constant potential can be applied. Inmeasurement, currents and voltages are controlled by using apotentiostat, digital multimeter, function generator, and the like.

EXAMPLE 3

The biological material detection element 5 shown in FIG. 11 was formedinto a current detection type interferon curative effect predicting DNAchip, and the following experiment was conducted.

First of all, a chromosome DNA was extracted from human leukocyte, a M×Agene fragment of about 100 bp was PCR-amplified by using a properprimer. The amplified fragment was heat-denatured. The resultantfragment was then introduced into the DNA chip. Note that a DNA probeassociated with SNP (single nucleotide polymorphism) existing in the M×Agene was immobilized in advance on an electrode 32 of the DNA chip.After the sample was introduced, it was left standing for two hr, andcleaning was done with a buffering agent. When a DNA binder (Hoechst33258) was made to act, it was found that an interferon curative effectcould be predicted even if the charge was not electrically controlled.However, it became clear that the results vary depending on the mannerin which the liquid flowed.

In contrast to this, it was found that a curative effect could bepredicted with high sensitivity and high accuracy without performing PCRamplification by the following operation. The introduced gene was movedto the peripheral portion while being concentrated by switching thepolarities of voltages applied to the respective electrodes, as shown inFIGS. 8A to 8C, and the gene was moved on the array of electrodes 32 byswitching the polarities of voltages applied to the respectiveelectrodes 32 while the gene was trapped on the electrode 32, as shownin FIGS. 7A to 7C. In addition, as described with reference to FIG. 12,the series of steps S3 to S6 is repeated a plural times.

Third Embodiment This embodiment relates to a charged material movingapparatus for moving a charged material such as a biological material,e.g., a gene or protein and, more particularly, to a charged materialmoving apparatus suitable for a biological material detection apparatus.This embodiment provides a charged material moving apparatus which canmove a charged material such as a biological material to make thematerial efficiently react with each ligand on a charged materialdetection element. In addition, there is provided a charged materialmoving apparatus which can concentrate a detection target chargedmaterial in a sample and attain an improvement in detection sensitivitywhen used for a biological material detection apparatus.

FIG. 14 is a sectional view showing the arrangement of a biologicalmaterial detection apparatus, to which a charged material movingapparatus is applied, according to a third embodiment of the presentinvention. A base 1 has a protruding element mount portion 2 on itscentral upper portion on which a biological material detection element 5(to be described in detail later) is mounted. The base 1 also has asample liquid passage hole 3 in the two sides in FIG. 14 and a sampleliquid outlet 4 in a central lower portion which communicates with thesample liquid passage hole 3.

An upper holder 6 and lower holder 7 are mounted on the base 1. Theupper and lower holders hold the biological material detection element 5mounted on the element mount portion 2 from both upper and lower sides,and mainly guide a sample liquid into the element 5 and the sampleliquid passing through the element 5 into the sample liquid passage hole3. The structures of the upper and lower holders 6 and 7 will bedescribed in detail later.

A sample liquid introduction portion 8 is formed in the central portionof the upper holder 6. A sample liquid supply pipe 10 is connected tothis sample liquid introduction portion 8. The sample liquid introducedfrom the sample liquid supply pipe 10 into the sample liquidintroduction portion 8 is guided onto the biological material detectionelement 5 and used for the detection of a biological material.Thereafter, the sample liquid is discharged outside from a sample liquidoutlet 4 through a sample liquid guide duct 9 formed by the lower andupper holders 6 and 7 and the sample liquid passage hole 3 formed in thebase 1.

A rectangular through hole 11 is formed in the upper holder 6. Thedistal end of a contact electrode 15 can be brought into contact with anelectrode serving as a working electrode on the biological materialdetection element 5 by inserting the contact electrode 15 via thethrough hole 11. By using this contact electrode 15, a biologicalmaterial detection signal can be extracted as an electrical signal suchas a current or potential signal.

FIG. 15 is a plan view showing the arrangement of the biologicalmaterial detection element 5. A sample liquid receiving portion 21formed from a recess portion is formed immediately under the sampleliquid introduction portion 8 on an element substrate 20 shown in FIG.14. A spiral sample liquid guide groove 22 having one end communicatingwith the sample liquid receiving portion 21 is formed on the elementsubstrate 20. A plurality of circular electrodes 23 functioning asworking electrodes are arrayed on the bottom portion of the guide groove22 along the spiral. A sample liquid discharge portion 24 is formed atthe other end of the guide groove 22 of the element substrate 20. Thissample liquid discharge portion 24 communicates with a sample liquidguide duct 9 shown in FIG. 14.

At least one type of specific detection ligand is immobilized to eachelectrode 23. That is, each electrode 23 also serves as a ligandimmobilizing portion. The ligand immobilized to each electrode 23 isselected from one of a gene, gene probe, protein, protein fragment,coenzyme, receptor, and sugar chain in accordance with the biologicalmaterial to be detected.

If different ligands are immobilized to the respective electrodes 23, aplurality of biological materials can be detected at once. In addition,if identical ligands are immobilized to the respective electrodes 23,many biological materials can be detected at once. If many electrodes 23(ligand immobilizing portions) are patterned on the element substrate 20in advance by photolithography, the productivity of biological materialdetection elements 5 improves.

The electrodes 23 are connected to electrode pads 25 via a multilayerinterconnection formed on the element substrate 20. A driving circuit 26for driving the electrodes 23 by applying predetermined voltages theretois connected to the electrode pads 25. The operation of this drivingcircuit 26 will be described in detail later.

The sample liquid supplied from the sample liquid supply pipe 10 in thismanner is guided by the upper holder 6 and lower holder 7 and introducedfrom the central portion into the biological material detection element5. After the sample liquid is uniformly supplied to the ligandimmobilizing portions formed on the peripheral portion of the element 5,the liquid is discharged from below. Detection of a biological materialin the sample liquid can therefore be done under a uniform condition.

If a biological material to be detected is a gene, a DNA probe isimmobilized as a ligand to the electrode 23. As is known, a DNA probe isa single stranded gene that reacts with a specific gene. If genes in asample liquid are converted into a single stranded gene in advance, onlya gene having a specific sequence in correspondence with the DNA probeimmobilized to the electrode 23 is trapped by the electrode 23.Subsequently, the DNA probe and the gene are complementarily bound toeach other (hybridization).

The above arrangement will be described in further detail. First of all,a substrate material used for the element substrate 20 is, but is notlimited to, for example, an inorganic insulating material such as glass,silica glass, alumina, sapphire, forsterite, silicon carbide, siliconoxide, or silicon nitride. Alternatively, one of the following organicmaterials can be used as a substrate material: polyethylene, ethylene,polypropylene, polyisobutylene, polymethylmethacrylate, polyethyleneterephthalate, unsaturated polyester, fluorine-containing resin,polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate,polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile,polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urearesin, epoxy resin, melamine resin, a styrene-acrylonitrile copolymer,an acrylonitrile-butadiene-styrene copolymer, silicone resin,polyphenylene oxide, polysulfone, and the like. In addition, ifbiological material detection is to be performed by the optical methodto be described later, a thin fiber film such as nylon or cellulose canbe used.

The electrode material to be used for the electrodes 23 is notspecifically limited. As a material for an electrode including ligandimmobilizing spot, when biological material detection is to be performedelectrochemically, for example, one of the following materials can beused: a single metal such as gold, gold alloy, silver, platinum,mercury, nickel, palladium, silicon, germanium, gallium, or tungsten,alloys each containing at least two of these metals, carbon such asgraphite or glassy carbon, oxides thereof, compounds thereof,semiconductor compounds such as silicon oxide, and various kinds ofsemiconductor devices such as a CCD, FET, and CMOS.

As a method of forming the electrodes 23, plating, printing, sputtering,vapor deposition, or the like can be used. As a vapor deposition method,one of a resistance-heating method, RF heating method, and electron beamheating method can be used. As a sputtering method, one of DC bipolarsputtering, bias sputtering, asymmetrical AC sputtering, gettersputtering, and RF sputtering can be used. For an electrode, anelectrolytic polymer film or conductive high polymer such as polypyrroleor polyaniline can be used.

An insulating material used for a thin insulating film that covers thesurfaces of the electrodes 23 is, but is not limited to, for example,photopolymer or photoresist material. As a photoresist material, anexposure photoresist, far ultraviolet photoresist, X-ray photoresist, orelectron beam photoresist may be used. A main material for an exposurephotoresist includes cyclized rubber, polycinnamic acid, and novolacresin. As a far ultraviolet photoresist, cyclized rubber, phenol resin,polymethylisopropenylketone (PMIPK), polymethylmethacrylate (PMMA), orthe like may be used. As an X-ray photoresist, a material written in“Thin Film Handbook” (Ohmsha, Ltd.) as well as a COP and metal acrylate.As an electron beam resist, a material written in “Thin Film Handbook”(Ohmsha, Ltd.) such as PMMA can be used. The resist to be used in thiscase preferably has a thickness of 10 nm or more and 1 mm or less.

The area of the electrode 23 serving as a working electrode can be madeuniform by covering the electrode 23 with a photoresist and performinglithography. With this process, the amounts of ligand such as DNA probeto be immobilized become uniform among the electrodes 23. This makes itpossible to perform biological material detection with excellentreproducibility. Conventionally, a resist material is generally removedin the end. If, however, the electrode 23 is used for the detection of agene immobilized to a DNA probe, the resist material can be used as partof the electrode 23 without being removed. In this case, a materialhaving high water resistance must be used as a resist material.

For the thin insulating film to be formed on the electrodes 23, amaterial other than photoresist materials can be used. For example,oxides, nitrides, and carbides of Si, Ti, Al, Zn, Pb, Cd, W, Mo, Cr, Ta,Ni, and the like and alloys thereof can be used. After a thin film isformed by sputtering, vapor deposition, CVD, or the like using one ofthese materials, the film is patterned by photolithography to formexposed electrode portions, thus controlling the area constant.

The driving operation of the driving circuit 26 will be described withreference to FIGS. 16A to 16C, 17A to 17C, 18A to 18C, and 19A to 19C.The driving circuit 26 applies voltages having predetermined polaritiesto the electrodes 23 to sequentially move a detection target biologicalmaterial in a sample liquid introduced to the sample liquid receivingportion 21 placed on the central portion on the biological materialdetection element 5 in the array direction of the electrodes 23.

When a detection target biological material is a gene, a sample liquidwhich is an aqueous solution of genes is supplied from the sample liquidsupply pipe 10 to the sample liquid receiving portion 21 on thebiological material detection element 5 via the sample liquidintroduction portion 8 and guided onto the electrode 23 via the guidegroove 22. The charge polarity of the gene is negative. FIGS. 16A to16C, 17A to 17C, 18A to 18C, and 19A to 19C show cases wherein drivingoperation is performed when a detection target biological material is agene. Each of FIGS. 16A to 16C, 17A to 17C, 18A to 18C, and 19A to 19Cshows changes in voltage applied state with respect to the electrodes 23over time. Assume that the voltage applied state (the polarity of avoltage) changes in the order of the suffixes A, B, and C attached tothe respective drawing numbers.

The examples of driving operation shown in FIGS. 16A to 16C will bedescribed. First of all, as shown in FIG. 16A, a positive voltage whosepolarity is opposite to the charge polarity of a gene is applied to anelectrode 23-1 nearest to the sample liquid receiving portion 21, and anegative voltage is then applied to an electrode 23-2 adjacent to theelectrode 23-1. The sample liquid supplied to the sample liquidreceiving portion 21 therefore moves onto the electrode 23-1 owing tothe electrostatic attracting force produced by the electrode 23-1 towhich the voltage whose polarity is opposite to the charge polarity ofthe gene is applied. In this case, if a slight negative pressure isapplied to the sample liquid, the sample liquid can move more quickly.Since a voltage having an opposite polarity to the charge polarity ofthe gene is applied to the electrode 23-2 adjacent to the electrode23-1, the gene is trapped on the electrode 23-1.

As shown in FIG. 16B, the polarity of the voltage applied to theelectrode 23-1 is then inverted to negative polarity, and the polarityof the voltage applied to the adjacent electrode 23-2 is also invertedto positive polarity. At this time, the gene in the sample liquidreceives electrostatic repulsive force from the electrode 23-1 to whichthe voltage having the same polarity as the charge polarity of the geneis applied and electrostatic attracting force from the electrode 23-2 towhich the voltage having the opposite polarity to the charge polarity isapplied. The gene therefore moves from the electrode 23-1 onto theelectrode 23-2 and trapped on the electrode 23-2.

As shown in FIG. 16C, the polarity of the voltage applied to theelectrode 23-2 is inverted to negative polarity, and the polarity of thevoltage applied to an adjacent electrode 23-3 is also inverted topositive polarity, thereby moving the gene in the sample liquid from theelectrode 23-2 onto the electrode 23-3 and trapping it on the electrode23-3.

Subsequently, the position of an electrode to which a voltage having thesame polarity as the charge polarity of the gene in the sample liquid isto be applied and the position of an electrode to which a voltage havingan opposite polarity to the charge polarity is to be applied aresequentially shifted in the same manner, thereby sequentially moving thegene onto the adjacent electrodes. In the process of this movement, ifthe specific gene held on a given electrode has a sequence complementaryto that of the ligand immobilized to the electrode, the gene and ligandreach and combine with each other. That is, hybridization occurs.

In this manner, in the biological material detection element 5, the genesequentially moves on the electrodes 23 along its array direction and isdischarged outside the element 5 from the electrode pads 24.

FIGS. 17A to 17C show another example of the driving operation of thedriving circuit 26 in this embodiment. Consider the example of drivingoperation shown in FIGS. 16A to 16C. Referring to FIG. 16B, for example,a positive voltage whose polarity is the same as the charge polarity ofthe gene is applied to only the electrode 23-1, of the electrodes 23,which is located on the opposite side to the electrode 23-2, on whichthe gene is trapped, in the moving direction of the gene. As shown inFIG. 17B, however, a positive voltage may also be applied to theelectrode 23-3 located along the moving direction of the gene. FIGS. 17Ato 17C respectively show voltage applied states at the same timings asthose in the case shown in FIGS. 16A to 16C. The first state shown inFIG. 17A is the same as that shown in FIG. 16A. In the case shown inFIGS. 17B and 17C, however, negative voltages are applied to theelectrodes located before and after, in the moving direction of thegene, the electrode to which a positive voltage is applied (on the twosides in the array direction of the electrodes 23).

With this operation, since negative voltages are applied to the twoelectrodes located before and after, in the moving direction of thegene, the gene, of the electrodes 23, on which the gene is trapped andto which a positive voltage is applied, the gene is confined on theelectrode on which the gene is trapped by the electrostatic repulsiveforce produced by the two electrodes, and the gene can be concentrated.This makes it possible to efficiently detect the gene. In addition, thegene need not be amplified or the degree of amplification is relaxed.

In the example of driving operation shown in FIGS. 18A to 18C, positivevoltages are applied to two adjacent electrodes of the electrodes 23,and a voltage having the opposite polarity is applied to an electrodeadjacent to the pair of electrodes. This operation is performed whileeach voltage application position is shifted by one electrode at a timein the array direction of the electrodes 23.

In the example of driving operation shown in FIGS. 19A to 19C, voltagesare simultaneously applied to all the electrodes 23. As in the caseshown in FIGS. 18A to 18C, positive voltages are applied to two adjacentelectrodes of the electrodes 23, and a voltage having the oppositepolarity is applied to an electrode adjacent to the pair of electrodes.This operation is performed while each voltage application position isshifted by one electrode at a time in the array direction of theelectrodes 23.

Letting n be the number of electrodes, of the electrodes 23, to whichpositive voltages whose polarity is opposite to the charge polarity ofthe gene are applied, m be the number of electrodes to which negativevoltages whose polarity is the same as the charge polarity are applied,and p be the number of electrodes by which the voltage applicationpositions are shifted when the polarities of applied voltages areswitched, n, m, and p can take arbitrary numbers equal to or larger thanone. Most simply, it suffices if n=m=p=1. In this case, the polarity ofan applied voltage is periodically and alternately switched betweenpositive polarity and negative polarity from the viewpoint of oneelectrode 23.

FIGS. 20A to 20C show the electrode arrangement and an example ofdriving operation of a biological material detection element accordingto still another embodiment of the present invention. As shown in FIGS.20A to 20C, according to this embodiment, a guide groove 32 is formed ona circumference centered on a sample liquid receiving portion 31 placedon the central portion of an element substrate in the biologicalmaterial detection element, and electrodes 33 to which ligands areimmobilized are arrayed on the bottom portion of this guide groove 32. Asample liquid supplied to the sample liquid receiving portion 31 isguided into the guide groove 32 and reaches the electrode 33.

In this arrangement, as in the example of driving operation shown inFIGS. 19A to 19C, a detection target biological material, e.g., a gene,in a sample liquid can be moved on the electrodes 33 along thecircumference by applying voltages to the electrodes 33 using a drivingcircuit (not shown) in the manner shown in FIGS. 20A to 20C.

First of all, as shown in FIG. 20A, positive voltages are applied to apair of adjacent electrodes, of the electrodes 33, which are enclosedwith the dotted line, and a negative voltage is applied to an electrodeadjacent to this pair of electrodes. After an elapse of a predeterminedunit time, as shown in FIG. 20B, the position of the pair of electrodesto which positive voltages are to be applied is shifted by oneelectrode, and the position of the electrode which is adjacent to thepair of electrodes and to which a negative voltage is to be applied isshifted by one electrode accordingly. When the predetermined unit timehas further elapsed, the position of the pair of electrodes to whichpositive voltages are to be applied and the position of the electrode towhich a negative voltage is to be applied are shifted by one electrode,as shown in FIG. 20C. Subsequently, such switching of the polarities ofapplied voltages will be done every unit time, i.e., in a predeterminedcycle. With this operation, a detection target biological material(e.g., a gene) in the sample liquid moves on the array of adjacentelectrodes 33 in the circumferential direction. This allows thedetection target biological material to uniformly and efficiently reactwith the ligand immobilized to each electrode 33.

In this manner, the detection target biological material in the sampleliquid introduced in the biological material detection element can besequentially moved on the electrodes 33 while being concentrated. Thatis, according to this embodiment, since the detection target biologicalmaterial is concentrated on the electrode 33 to which the ligand isimmobilized, there is no need to amplify the detection target biologicalmaterial by a gene amplification method such as the PCR method as in theprior art, and the detection target biological material and ligand canbe made to efficiently reach with each other. This improves thedetection efficiency.

FIGS. 21A and 21B are a plan view and sectional view showing stillanother arrangement of a biological material detection apparatusaccording to the third embodiment.

In the biological material detection apparatus according to thisembodiment, a sample liquid receiving portion 41 is formed in the centerof a protruding portion 40A formed on the central portion of a base 40,and a recess portion 42 is so formed as to be centered on the sampleliquid receiving portion 41. In addition, a plurality of fan-shapedelectrodes 43 are formed within the recess portion 42 so as to radiallyextend from the sample liquid receiving portion 41. Ligands areimmobilized to the electrodes 43. In addition, the base 40 has a sampleliquid passage hole 44 which guides the sample liquid that has passedthrough the biological material detection element to the sample liquidoutlet.

In this biological material detection apparatus as well, by driving theelectrodes 43 using a driving circuit (not shown) in the same manner asin the case described with reference to FIGS. 20A to 20C, the detectiontarget biological material is sequentially moved in the circumferentialdirection in which the electrodes 43 are arrayed while beingconcentrated, and can be made to efficiently react with the ligand.

FIG. 22 is a plan view showing the arrangement of a biological materialdetection element according to a modification of the third embodiment.

A sample liquid receiving portion 51 and sample liquid discharge portion54 are arranged near two opposite corners on an element substrate 50. Azigzag sample liquid guide groove 52 is formed between the sample liquidreceiving portion 51 and the sample liquid discharge portion 54.Electrodes 53 to which ligands are immobilized are arranged in thissample liquid guide groove 52.

In the use of the biological material detection element having thisarrangement as well, a detection target biological material issequentially moved in the circumferential direction in which theelectrodes 53 are arrayed by performing the same driving operation asthat described with reference to FIGS. 16A to 16C, 18A to 18C, 19A to19C, and 20A to 20C using a driving circuit (not shown). In addition,since the detection target biological material is concentrated duringmovement, the ligand can be made to efficiency react with the material.

Fourth Embodiment

FIG. 23 shows the schematic arrangement of a biological materialprocessing apparatus having a plurality of reaction steps according to afourth embodiment of the present invention.

In this embodiment, a plurality of reaction chambers 61A to 61F arearranged on a substrate 60. These reaction chambers 61A to 61Fcommunicate with each other via sample liquid guide grooves 62, asneeded. Electrodes 63 are arrayed in the sample liquid guide grooves 62.For example, ligands may be immobilized to the electrodes 63.

In this biological material processing apparatus as well, a detectiontarget biological material is sequentially moved in the circumferentialdirection in which the electrodes 63 are arrayed by performing the samedriving operation as that described with reference to FIGS. 16A to 16C,18A to 18C, 19A to 19C, and 20A to 20C using a driving circuit (notshown). In addition, since the detection target biological material isconcentrated during movement, reactive processing of the detectiontarget biological material can be efficiently done in the reactionchambers 61A to 61F.

Fifth Embodiment FIG. 24 is a view showing a micellar structure used ina biological material processing apparatus, to which a charged materialmoving apparatus is applied, according to a fifth embodiment of thepresent invention. This embodiment is an apparatus used to process abiological material having no charge. In this apparatus, a micellarstructure is formed by using, for example, a surfactant.

A micellar structure is a structure in which an uncharged material iscovered with a micelle to be forcibly charged. Referring to FIG. 24, abiological material is charged to negative polarity. Even when abiological material forcibly charged by a micellar structure in thismanner is to be moved, the biological material moving apparatus orbiological material processing apparatus having the arrangementdescribed in each of the above embodiments can be used.

A sample to be processed by the biological material detection element orbiological material detection apparatus using the biological materialmoving apparatus according to the embodiment is, but is not limited to,for example, blood, blood serum, leukocyte, urine, stool, sperm, saliva,tissue, cultured cell, expectoration, and the like. If, a detectiontarget biological material is a gene, the gene is extracted from thesesamples. The extraction method may be, but is not limited to aliquid-liquid extraction method such as a phenol-chloroform method or asolid liquid extraction method using a carrier. Alternatively, acommercially available nucleic acid extraction method such as QIAamp(available from QIAGEN), SUMAI test (available from Sumitomo MetalIndustries, Ltd.) can be used.

The gene sample solution extracted in this manner is then introducedonto the biological material detection element (DNA chip) described inthe above embodiment, and a hybridization reaction is caused on anelectrode to which a DNA probe as a ligand is immobilized. A bufferingsolution that falls within the range of ion intensities of 0.01 to 5 andthe range of pH5 to pH10 is used as a reaction solution. A hybridizationaccelerating agent such as dextran sulfate, salmon sperm DNA, bovinethymus DNA, EDTA, or surfactant can be added to this solution, asneeded. The extracted sample gene is added to this solution. Thesolution must be heat-denatured at 90° C. or more before introductioninto the biological material detection element. An unreacted sample genecan be recovered from the sample liquid outlet 4 to be introduced intothe biological material detection element again, as needed.

The extracted gene can be detected by marking in advance it with afluorescent dye such as FITC, Cy3, Cy5, or rhodamine, an enzyme such asbiotin, hapten, oxidase, or phosphatase, or an electrochemically activematerial such as ferrocene or quinones, or by using a second probemarked with such a material. If the gene is marked with a fluorescentdye, it can be optically detected.

When the gene is to be detected by using an electrochemically active DNAbinder, detection is done by the following procedure.

A DNA binder which selectively combines with a double stranded DNAportion is made to react with the double stranded DNA portion formed onthe surface of an electrode (working electrode) to which a DNA probe isimmobilized, thereby performing electrochemical measurement. The DNAbinder to be used in this case is, but is not limited to, for example,Hoechst 33258, acridine orange, quinaccrine, downomycin,metallointercalator, bis-intercalator such as bis-acridine,tris-intercalator, or polyintercalator. In addition, such anintercalator can be modified in advance by an electrochemically activemetal complex such as ferrocene or viologen.

Although the concentration of a DNA binder varies depending on the typeof material, the material is generally used within the range of 1 ng/mLto 1 mg/mL. In this case, a buffering agent within the range of ionintensities of 0.001 to 5 and the range of pH5 to pH10 is used.

After an electrode serving as a working electrode and a DNA binder aremade to react with each other, cleaning is performed, andelectrochemical measurement is performed. Electrochemical measurement isperformed by a triple electrode type including a reference electrode,counterelectrode, and working electrode or a double electrode typeincluding a counterelectrode and working electrode. In measurement, apotential equal to or higher than a potential at which the DNA binderreacts is applied, and a reaction current value originating from the DNAbinder is measured. In this case, the potential is swept at a constantvelocity or pulses or a constant potential can be applied. Inmeasurement, currents and voltages are controlled by using apotentiostat, digital multimeter, function generator, and the like.

EXAMPLE 4

The biological material detection element described with reference toFIGS. 19A to 19C was formed into an interferon curative effectpredicting DNA chip and the following experiment was conducted.

First of all, a chromosome DNA was extracted from human leukocyte, a M×Agene fragment of about 100 bp was PCR-amplified by using a properprimer. The amplified fragment was heat-denatured. The resultantfragment was then introduced into the DNA chip. Note that a DNA probeassociated with SNP (single nucleotide polymorphism) existing in the M×Agene was immobilized in advance on an electrode 23 of the DNA chip.After the sample was introduced, the electrodes 23 were driven. After anexcess sample liquid was finally observed, cleaning was done with abuffering agent. When a DNA binder (Hoechst 33258) was made to act, itwas found that an interferon curative effect could be predicted.

In this case, it was found that when the introduced gene was moved tothe peripheral portion while being concentrated by switching thepolarities of voltages applied to the electrodes 23 in the same manneras described with reference to FIGS. 19A to 19C, and the gene was movedon the array of electrodes 32 by switching the polarities of voltagesapplied to the respective electrodes 23 while the gene was trapped onthe electrode 23, a curative effect could be accurately predictedwithout performing PCR amplification.

EXAMPLE 5

The biological material detection element described with reference toFIG. 22 was formed into a tumor marker detection chip, and the followingexperiment was conducted.

To form a tumor marker detection chip, antibodies for various humantumor markers are immobilized to the surfaces of electrodes 53. In orderto prevent nonspecific adsorption, 1% bovine serum albumin was made toact. When the charges of the electrodes were changed by operationsimilar to that in Example 1, and tumor markers were detected by usingthe human serum as a sample, it was found that the markers could bedetected with high reproducibility and high sensitivity on the order of0.1 ng/mL. In this case, an antibody marked with horseradish peroxidasewas used as the second antibody, and a framework for luminescencedetection was supplied lastly.

The above embodiment has exemplified only the biological material movingapparatus. However, the present invention is not limited to thebiological material moving apparatus and can be applied to allapparatuses for moving charged materials having predetermined chargepolarities.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1-17. (canceled)
 18. A biological material moving apparatus for moving acharged biological material contained in a sample liquid, comprising: abiological material detection element having a substrate and a pluralityof electrodes which are arrayed at predetermined intervals along acircumferential direction on the substrate and to which ligands thatreact with predetermined biological materials are respectivelyimmobilized; a sample liquid introduction part to introduce the sampleliquid to a central portion of the array of the electrodes on thesubstrate; a sample liquid moving mechanism to move the sample liquidintroduced to the central portion on the substrate by the sample liquidintroduction part radially toward the electrodes by an electrostaticattracting force produced by the electrodes; and a control deviceconfigured to control a process which is performed repeatedly, theprocess including (a) supplying an intercalating agent onto thebiological material detection element, (b) measuring an electrochemicalsignal from the intercalating agent which is based on a reaction betweenthe charged biological material and the ligand, and (c) removing theintercalating agent adhering to the ligand.
 19. A biological materialmoving apparatus to which a sample liquid containing a chargedbiological material is introduced, and which moves the biologicalmaterial, comprising: a biological material detection element having asubstrate, at least one first electrode placed at a position on thesubstrate to which the sample liquid is introduced, and a plurality ofsecond electrodes which are arrayed at predetermined intervals aroundthe first electrode on the substrate along a circumferential direction,and to which ligands that react with predetermined biological materialsare respectively immobilized; a driving circuit which drives thebiological material detection element by performing a driving operationof applying a voltage having the same polarity as a charge polarity ofthe biological material to the first electrode, and applying a voltagehaving an opposite polarity to the charge polarity to at least some ofthe second electrodes, the first electrode and the second electrodeseach producing an electrostatic field by which the charged biologicalmaterial is moved toward the second electrodes; and a control deviceconfigured to control a process which is performed repeatedly, theprocess including (a) supplying an intercalating agent onto thebiological material detection element, (b) measuring an electrochemicalsignal from the intercalating agent which is based on a reaction betweenthe charged biological material and the ligand, and (c) removing theintercalating agent adhering to the ligand.
 20. The apparatus accordingto claim 19, wherein the driving circuit further performs a drivingoperation of applying a voltage having an opposite polarity to thecharge polarity to some of the second electrodes in the circumferentialdirection and applying a voltage having the same polarity as the chargepolarity to some other electrodes of the second electrodes, whilesequentially changing positions of electrodes to which voltages havingthe opposite polarity and same polarity are to be applied.
 21. Theapparatus according to claim 19, wherein the biological materialdetection element further includes at least two annular electrodesconcentrically arranged between the first electrode and the array of thesecond electrodes